Interleukin 31 receptor alpha augments muscarinic acetylcholine receptor 3-driven calcium signaling and airway hyperresponsiveness in asthma

Asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR), inflammation, and goblet cell hyperplasia. Both Th1 and Th2 cytokines, including IFN-γ, IL-4, and IL-13 have been shown to induce asthma; however, the underlying mechanisms remain unclear. We observed a significant increase in the expression of IL-31RA, but not its cognate ligand IL-31 during allergic asthma. In support of this, IFN-γ and Th2 cytokines, IL-4 and IL-13, upregulated IL-31RA but not IL-31 in airway smooth muscle cells (ASMC). Importantly, the loss of IL-31RA attenuated AHR but had no effects on inflammation and goblet cell hyperplasia in allergic asthma or mice treated with IL-13 or IFN-γ. Mechanistically, we demonstrate that IL-31RA functions as a positive regulator of muscarinic acetylcholine receptor 3 expression and calcium signaling in ASMC. Together, these results identified a novel role for IL-31RA in AHR distinct from airway inflammation and goblet cell hyperplasia in asthma.


INTRODUCTION
Asthma is a common chronic inflammatory disease that affects more than 300 million people worldwide. Asthma is associated with significant health and economic burdens and impairs both quality of life and productivity 1,2 . Despite advances in understanding the disease physiopathology and the development of anti-inflammatory therapeutic interventions, the incidence of asthma, including uncontrolled and severe forms, continues to increase. The disease is characterized by episodes of airflow obstruction that are associated with high mortality when severe and unreversed 3 . The primary clinical manifestations in the pathophysiology of chronic asthma include airway hyperresponsiveness (AHR), inflammation, goblet cell hyperplasia with mucus production, and fibrotic airway remodeling 4 . AHR is one of the cardinal features of asthma, but AHR can also be present in other airway disorders such 7 effect of IL-31RA deficiency on allergic inflammatory response in airways. We further investigated whether the loss of IL-31RA had any effect on the infiltration of specific immune cell types into the airspaces by differential counting of the immune cell types in BAL.
Consistent with the above findings, we observed no significant differences in the number of eosinophils, macrophages, lymphocytes, and neutrophils between wild-type and IL-31RA -/mice challenged with either saline or HDM (Fig 2C).
Accumulation of mucus-secreting goblet cells or goblet cell hyperplasia is another important hallmark of allergic asthma observed in both human and animal models of allergic asthma [43][44][45] . To determine whether there were differences in goblet cell hyperplasia, we performed alcian blue periodic acid-Schiff (ABPAS) staining to detect mucus-producing goblet cells in lung sections. Both wild-type and IL-31RA -/mice displayed increased goblet cell hyperplasia when challenged with HDM compared to unchallenged naïve animals (Fig 2D and   2E). These findings suggest that IL-31RA deficiency contributes to the development of AHR but has no significant effect on airway inflammation and mucus production in HDM-induced allergic asthma.
IL-31RA functions as a negative regulator of Th2 responses in the lungs 46 However, the role of IL-31RA in the development of Th2 responses during allergic asthma remains unclear. To determine whether there are alterations in the expression of asthma-associated genes in the absence of IL-31RA, we measured the transcript levels of Th2 cytokines, inflammatory cytokines, Th2 responses, and goblet cell hyperplasia-associated genes. As expected, we observed a significant increase in Th2 cytokine gene expression, including IL-4, IL-5, and IL-13, in wild-type mice challenged with HDM compared to that with saline ( Fig   3A). However, HDM-induced increase in Th2 cytokine expression was similar in the lungs of wild-type and IL-31RA -/mice, suggesting that the absence of IL-31RA had no effect on HDMinduced Th2 cytokine expression. Similarly, the loss of IL-31RA had no effect on the expression of chemokine and cytokine genes (CCL11, CCL24, and IL-10) and Th2 responseassociated genes, including ARG1, CHI3L3, and FIZZ1 (Fig 3B & 3C). Consistent with ABPAS staining, we observed no effect of IL-31RA deficiency on HDM-induced expression of GOB5 and MUC5AC (Fig 3D). IL-6 and oncostatin M (OSM), together with IL-31, are members of the IL-6 family of cytokines known to regulate AHR in allergic asthma [47][48][49] . Thus, we explored the levels of OSM and IL-6 in the lungs of wild-type and IL-31RA -/mice ( Fig   S1). We observed no significant differences in the expression of IL-6 and OSM between wildtype and IL-31RA -/mice treated with either saline or HDM, suggesting that the absence of IL-31RA did not affect the expression of OSM and IL-6.

IL-31RA is essential to induce AHR during SEA-induced allergic asthma
To further establish the role of IL-31RA in allergic asthma, we used an alternative mouse model of SEA-induced allergic asthma. Similar to HDM, extract of Schistosoma mansoni is a potent inducer of AHR, airway inflammation, and Th2 immune responses even without the use of an adjuvant 28, 50,51 . As shown in the schematic, wild-type and IL-31RA -/mice were sensitized twice and consecutively challenged with SEA to induce allergic asthma ( Fig 4A). The absence of IL-31RA gene expression was confirmed by measuring transcripts in the lungs of mice challenged with SEA or saline, and no IL-31RA expression was detected in IL31RA -/mice ( Fig 4B). Consistent with the findings using the HDM model, sensitization and challenge with SEA resulted in a significant increase in the gene expression of IL-31RA but not IL-31 in the wild-type mice (Fig 4B & 4C).
To assess whether the loss of IL-31RA attenuates SEA-induced AHR, we measured MCh-induced AHR in both wild-type and IL-31RA -/mice sensitized and challenged with either saline or SEA (Fig 4D). Loss of IL-31RA was sufficient to attenuate MCh-induced AHR in SEA-challenged mice (Fig 4D). To evaluate the changes in airway inflammation, lung sections of wild-type and IL31RA -/mice challenged with SEA or saline were stained with H&E. As observed in the HDM model, SEA challenge induced robust airway inflammation with immune cell infiltration in both wild-type and IL-31RA -/mice ( Fig 4E). Similarly, the airways of IL-31RA -/mice that were challenged with SEA showed an accumulation of mucusproducing goblet cells similar to wild-type mice, as seen in ABPAS-stained lung sections ( Fig   4F).
To determine whether IL-31RA-induced AHR is regulated by asthma-associated gene networks, we measured the expression of Th1 and Th2 cytokines. The expression of IFN-γ, IL-4, and IL-5 was elevated in both wild-type and IL-31RA -/mice following sensitization and challenge with SEA ( Fig 5A). Similarly, the transcripts of genes associated with inflammation (CCL11, CCL24, and IL-10) and Th2 responses (ARG1, CHI3L3, and FIZZ1) remained elevated in IL-31RA -/mice, and no differences were observed when compared to wild-type mice treated with SEA (Fig 5B & 5C). The gene transcripts associated with goblet cell hyperplasia were elevated in both wild-type and IL-31RA -/mice treated with SEA ( Fig 5D).
In summary, in the two alternative mouse models of allergic asthma, the absence of IL-31RA altered AHR but did not appear to affect airway inflammation, Th2 responses, or goblet cell hyperplasia.

IL-31 is dispensable to induce AHR, inflammation, and Th2 responses
IL-31RA is a cognate binding receptor of IL-31 that recruits OSMRβ to form a high-affinity binding receptor complex that signals through the JAK/STAT pathway 29,30 . Previous studies have demonstrated that increased IL-31 signaling through IL-31RA can result in uncontrolled inflammation and tissue remodeling in multiple tissues, including the lungs and skin 25,26 .
However, we observed no significant increase in IL-31 levels in the lungs during HDM-and SEA-induced allergic asthma. To assess the potential pathological effects of IL-31 in asthma, the lungs of wild-type mice were intratracheally treated with saline or IL-31, and changes in AHR and inflammation were assessed ( Fig 6A). Notably, we observed no significant changes in AHR between saline and IL-31 treated wild-type mice (Fig 6B). Similarly, we observed no significant changes in tissue inflammation as assessed by H&E-stained lung sections of saline and IL-31 treated wild-type mice (Fig 6C). To confirm the signaling effects of IL-31, we measured the expression of the IL-31-driven gene suppressor of cytokine signaling 3 (SOCS3) in the lungs of mice treated with saline and IL-31. Previously published studies have shown that IL-31 upregulates SOCS3 52 ; similarly, the expression of SOCS3 was significantly upregulated in lungs of IL-31-treated mice compared to the saline-treated mice ( Fig 6D). To determine the effects of IL-31 on the expression of asthma-associated genes, we quantified the expression of genes associated with inflammation (IFN-γ, TNF-α, IL-6, and IL-17) and Th2 responses (IL-4, IL-13, ARG1, MUC4, and MUC5AC). Notably, we observed no significant changes in the expression of genes associated with either inflammation or Th2 responses ( Fig   6E and 6F).
Furthermore, we evaluated airway contractility induced by IL-31 using PCLS. As demonstrated in previous studies, we observed a significant increase in the contractility of airways of wild-type mice treated with IL-13 compared to saline-treated mice in a dosedependent manner with MCh ( Fig S2). Conversely, we observed no significant effect of IL-31 on the contractility of wild-type airways exposed to MCh in comparison to media ( Fig 6G).
Furthermore, we observed no significant changes in the kinetics of contraction of collagen gels embedded with ASMC, which were treated with either media alone or IL-31. However, we observed a significant increase in the contraction of collagen gels embedded with ASMC and treated with IL-13 compared to that with media alone (Fig 6H). Thus, in contrast to IL-31RA, the results suggest that IL-31 is not involved in modifying AHR, inflammation, and Th2 responses in the lungs; the reduced AHR observed in the absence of IL-31RA could be due to other alternative mechanisms that need to be identified.
Th2 cytokines upregulate IL-31RA to induce AHR with no effect on inflammation Previous studies from our lab and others have shown that both IL-4 and IL-13 induce IL-31RA expression in macrophages and lung tissues via the type II IL-4 receptor and STAT6 signaling 38 . To determine whether IL-4 or IL-13 can induce IL-31RA expression in ASMC, we treated ASMC with increasing doses of IL-4 or IL-13 and quantified the expression of IL31RA.
Consistent with our earlier findings in macrophages, the expression of IL-31RA significantly increased in ASMC treated with IL-4 or IL-13 compared to that with media ( Fig 7A & 7 B).
IL-13 is a potent inducer of AHR, inflammation, and goblet cell hyperplasia in mice 18 . To determine whether IL-13-induced AHR is IL-31RA dependent, we treated both wild-type and IL-31RA -/mice with IL-13 and assessed AHR and other pathological and molecular changes that are relevant to asthma. Notably, the loss of IL-31RA was sufficient to attenuate IL-13induced AHR compared to wild-type mice exposed to increasing doses of MCh ( Fig 7C).
Similarly, the contraction of collagen gels embedded with ASMC from IL-31RA -/mice significantly reduced compared to wild-type mice with IL-13 treatment ( Fig 7D). In contrast, H&E staining of lung sections suggested that the loss of IL-31RA had no effect on IL-13driven airway inflammation in wild-type and IL31RA -/mice ( Fig 7E). Quantitative assessment of inflammatory chemokines and cytokines suggested that the loss of IL-31RA had no effect on the expression of CCL11, CCL24, and IL-17 ( Fig 7F). Similarly, we observed no defects in the expression of Th2 cytokines (IL-4 and IL-5) or genes associated with Th2 responses (ARG1, CHI3L3, and FIZZ1) in IL-31RA -/mice compared to wild-type mice treated with IL-12 To evaluate whether IL-13-induced goblet cell hyperplasia was altered in the absence of IL-31RA, we assessed the accumulation of goblet cells in the airways and quantified the transcripts associated with goblet cell hyperplasia. As shown in figure 7I, goblet cell accumulation was similar between wild-type and IL31RA -/mice treated with IL-13. In support of this, the expression of GOB5 and MUC5AC remained similar between IL-13 treated wildtype and IL31RA -/mice ( Fig 7J). Thus, IL-31RA expression is essential to mediate IL-13induced AHR but dispensable for inflammation and goblet cell hyperplasia.

IFN-γ upregulates IL-31RA to induce AHR with no effects on inflammation
IFN-γ is a key Th1 cytokine implicated in elevated AHR in patients with severe asthma and those with mixed Th1/Th2 cytokine phenotypes 12,53 . To determine whether IFN-γ induces the expression of IL-31RA in ASMC, we isolated ASMC from the tracheas of wild-type mice and treated them with increasing doses of IFN-γ. The expression of IL-31RA is significantly increased by IFN-γ in a dose dependent manner (Fig 8A). A recent study suggested that IL-31 can also upregulate the expression of IL-31RA in human sensory neurons 54 . However, we observed no significant effect of IL-31 on the expression of IL-31RA in ASMC (Fig 8B). To determine the effects of IFN-γ on asthma phenotypes, we treated wild-type mice with saline or IFN-γ and measured the changes in AHR, inflammation, and goblet cell hyperplasia. We observed a significant increase in AHR in wild-type mice treated with IFN-γ compared to that with saline ( Fig 8C). In addition, we observed a significant increase in peri-bronchial inflammation in wild-type mice treated with IFN-γ compared to that with saline ( Fig 8D). To establish the quantitative gene expression changes induced by IFN-γ, we measured IRF1, IRF7, and STAT1 and observed a significant increase in the expression of IFN-γ-specific genes in the lungs (Fig S3).
To determine the effects of IFN-γ on asthma phenotypes, we measured the expression of genes associated with inflammation and Th2 responses. In support of the substantial inflammation observed after IFN-γ treatment, we observed a significant increase in CCL11 and IL-17 expression ( Fig 8E). As anticipated, IFN-γ treatment resulted in negative regulation of Th2 cytokines and Th2 response-associated gene expression, including IL-4, IL-5, CCL24, ARG1, and CHI3L3, but had no effect on the expression of FIZZ1, which was significantly increased compared to the saline group (Fig 8F and G). Similar to IL-13, IFN-γ was able to induce the accumulation of goblet cells in the airways with increased expression of GOB5 and MUC5AC (Fig 8H and 8I). Despite the reduced expression of several Th2-associated asthma genes, IFN-γ treatment induced AHR, inflammation, and goblet cell hyperplasia.
To determine whether the expression of IL-31RA was critical for IFN-γ-induced AHR, inflammation, and goblet cell hyperplasia, we intratracheally treated both wild-type and IL31RA -/mice with IFN-γ. Notably, IFN-γ-induced AHR was significantly attenuated in IL31RA -/mice compared to that in wild-type mice ( Fig 9A and Fig S4). However, as noted with IL-13, increases in peribronchial inflammation and the expression of inflammatory cytokine genes, such as CCL11, CCL24, and IL-17, were similar between wild-type and IL-31RA -/mice treated with IFN-γ ( Fig 9B and 9C). Furthermore, we observed no significant differences in the expression of Th2 cytokine genes (IL-4 and IL-5) or the expression of genes associated with Th2 responses, including ARG1, CHI3L3, and FIZZ1 (Fig 9D and 9E).
To evaluate the effects of IL-31RA deficiency on IFN-γ-induced goblet cell hyperplasia, we performed ABPAS staining of lung sections from wild-type and IL-31RA -/mice and quantified GOB5 and MUC5AC expression. Analysis of ABPAS-stained lung sections suggested no change in the number of goblet cells that accumulated in the airways of wild-type and IL31RA -/-mice treated with IFNγ ( Fig 9F). This finding was further substantiated by quantitative PCR data, which showed no quantitative differences in the expression of GOB5 and MUC5AC with the loss of IL-31RA ( Fig 9G). Nevertheless, our in vivo studies demonstrated that despite the development of substantial inflammation and mucus hypersecretion, IL-31RA -/mice showed attenuated AHR in response to either IL-13 or IFN-γ.
Overall, our new findings convincingly demonstrate that in allergic asthma, IL-31RA, which is induced by both Th1 and Th2, is critically required for the development of AHR but not inflammation and mucus secretion.

IL-31RA augments CHRM3-driven calcium signaling in ASMC
Muscarinic acetylcholine receptors (CHRMs) are predominantly expressed by structural cells such as smooth muscle cells of airways and play a major role in triggering contraction of airways and AHR 55,56 . To evaluate mechanisms by which IL-31RA induces AHR, we measured the transcripts of five major receptor subtypes including CHRM1, CHRM2, CHRM3, CHRM4 and CHRM5 in the lungs of wild-type and IL-31RA -/mice. Quantification of the lung transcripts suggest no significant differences in the transcript levels of CHRMs in the lungs of IL-31RA -/mice compared to wild-type mice ( Fig 10A). CHRM3 is a dominant receptor subtype expressed in ASMC and coupled to the Gq/11 family of G proteins to induce calcium signaling and the contraction of ASMC in asthma 55,57 . Therefore, we evaluated the changes in the transcripts of CHRM3 by IL-4 and IL-31 in ASMC of wild-type and IL-31RA -/mice. Neither IL-4 and IL-31 had a significant effect on the expression of CHRM3 either in the presence or absence of IL-31RA suggesting other mechanisms might be involved in IL-31RA-driven contraction of ASMC (Fig 10B and 10C). To determine post-transcriptional regulation of CHRM3, we measured the protein levels of CHRM3 in AMSC isolated from wild-type and IL-31RA -/mice. Notably, we observed a significant decrease in the protein levels of CHRM3 in ASMC isolated from IL-31RA -/mice compared to wild-type mice ( Fig   10D). The decrease in CHRM3 protein with no changes in the transcript levels in the absence of IL-31RA may suggest a post-transcriptional stabilization of CHRM3 that may involve a physical interaction between IL-31RA and CHRM3 in ASMC. To identify the complex formation between IL-31RA and CHRM3, we used in situ proximity ligation assay (PLA) and measured the complex formation between IL-31RA and CHRM3 in HEK293T cells overexpressing both IL-31RA and CHRM3. We generated overexpression plasmids and our western blot analysis of HEK293T cell lysates show overexpression of IL-31RA and CHRM3 in HEK293T cells ( Fig S5). Importantly, we observed bright fluorescent signals corresponding to the IL-31RA-CHRM3 complex formation in HEK293T cells overexpressing IL-31RA and CHRM3 compared to control cells ( Fig 10E). By fluorescently labeling plasma membrane with cholera toxin, we demonstrated the colocalization of PLA puncta with the plasma membrane in HEK293T cells overexpressing IL-31RA and CHRM3 compared to control cells ( Fig S6).
These results demonstrated the specificity of the PLA methodology to detect IL-31RA-CHRM3 complex formation in HEK293T cells overexpressing both IL-31RA and CHRM3 compared to control cells. Next, we assessed whether IL-31RA physically interacts with CHRM3 to augment smooth muscle cell contractility. To assess this, we performed coimmunoprecipitation studies with HEK293T cells transfected with plasmid overexpressing Cterminal FLAG-tagged IL-31RA as these cells naturally express low levels of CHRM3. When we immunoprecipitated FLAG-tagged IL-31RA from cell extracts with antibody against FLAG-tag, endogenous CHRM3 protein was co-purified, as assessed by western blot analysis ( Fig 10F). We could not coimmunoprecipitate endogenous CHRM3, a negative control with IgG control or cell lysates from HEK293T cell transfected with control plasmid. Because of the physical interaction between the CHRM3 and IL-31RA, we hypothesized that IL-31RA augments CHRM3-driven calcium signaling. Therefore, we assessed the gain-of-function effects of IL-31RA on CHRM3-driven calcium signaling by carbachol in HEK293T cells.
Carbachol treatment in HEK293T cells transfected with control plasmids resulted in elevated intracellular calcium flux ( Fig 10G). Notably, overexpression of IL-31RA was sufficient to augment calcium release similar to the levels observed with CHRM3 overexpression. This increase in intracellular calcium release was further elevated in HEK293T cells that coexpressed both IL-31RA and CHRM3 which may suggest a cooperation between IL-31RA and CHRM3 to augment carbachol-induced calcium flux. However, calcium-dependent phosphorylation of myosin light chain (MLC) is a terminal event in the contraction of ASMC 58 . Therefore, we measured carbachol-induced phosphorylation of MLC in HEK293T cells overexpressing IL-31RA compared to control HEK293T cells. Importantly, overexpression of IL-31RA alone was sufficient to augment the levels of carbachol-induced MLC phosphorylation that involved in smooth muscle contraction ( Fig 10H). To further demonstrate the positive regulation of MLC phosphorylation by the IL-31RA-CHRM3 axis, we measured carbachol-induced phosphorylation of MLC in ASMC isolated from wild-type and IL-31RA -/mice. Consistent with reduced CHRM3 expression in IL-31RA deficient ASMC, we observed a significant decrease in carbachol-induced MLC phosphorylation in ASMC isolated from IL-31RA -/mice compared to wild-type mice (Fig 10I). These results suggest that IL-31RA functions as a positive regulator of CHRM3 and associated carbachol-induced calcium signaling to augment the contractility of ASMC in asthma.

DISCUSSION
In this study, we investigated the pathophysiological role of the IL-31/IL-31RA axis using two complementary mouse models of allergic asthma. Our Th1/Th2 cytokine intratracheal instillation studies using wild-type and IL-31RA knockout mice revealed an uncoupling of AHR from other airway pathologies typically underlying asthma, including inflammation and goblet cell hyperplasia. Consistent with dominant role for IL-31RA but not IL-31 in asthma pathogenesis in these models, only the receptor for IL-31 was elevated in wild-type mice exposed to allergens, including HDM and SEA, and administration of IL-31 had 1) limited or no effect on MCh-induced AHR, inflammation, and goblet cell hyperplasia and 2) no significant changes in the contraction of airways in the PCLS or collagen gel-embedded ASMCs.
Our findings suggest that the expression of IL-31RA is not essential for both Th1 and Th2 cytokine-induced inflammation and goblet cell hyperplasia. However, this apparently limited role of IL-31RA is consistent with a recently published study in which neutralization of IL-31 had no effect on airway inflammation in wild-type mice that were sensitized and We also provide a novel finding that IL-31RA functions as a positive regulator of CHRM3-driven calcium signaling and contractility of ASMC. This is the first study, to our knowledge to show that the IL-31RA-CHRM3 axis induces the phosphorylation of MLC in ASMC. CHRM3 is a key muscarinic receptor expressed by smooth muscle cells in the airways and plays a major role in the contractility of smooth muscle cells in response to muscarinic ligands such as acetylcholine and methacholine 56,58 . CHRM3 is a member of class A GPCR that can mediate ASMC contraction through both calcium-dependent and calcium-independent mechanisms 55,58 . The calcium-dependent smooth muscle cell contractility is dependent on the activation of a subunit of Gq and the release of inositol 1,4,5-triphosphate by phospholipase C 69 . Our studies using both the loss of function and gain-of-function studies suggest that IL-31RA augments CHRM3-driven calcium release and phosphorylation of MLC. Notably, we observed a possible physical interaction between IL-31RA and CHRM3 that may contribute to enhanced calcium signaling and contractility of ASMC through stabilizing CHRM3 protein. It is possible other mechanisms that involve stabilizing CHRM3 or other proteins in macromolecular complexes that involved in calcium-dependent and calcium-independent ASMC contraction 55,58 . How these observations including the physical interaction between IL-31RA and CHRM3 are linked to elevated CHRM3-driven signaling remains unknown.
Further studies are required to determine the validity of the hypothesis that IL-31RA stabilizes CHRM3 to augment intracellular calcium signaling in the contraction of ASMC and whether it extends to other CHRMs. Nevertheless, given the robust increases in CHRM3-driven calcium signaling, and ASMC contraction, it is easy to envision the scope of the IL-31RA-CHRM3 axis in inducing AHR in asthma. Our findings provide evidence of the potential therapeutic benefits of targeting IL-31RA, which is downstream of both Th1 and Th2 cytokines, to attenuate AHR.
This hypothesis is important for further investigation because therapeutic antibodies targeting IL-31RA are currently in multiple phase II clinical trials for atopic dermatitis 70 . Given these findings, it is important to determine whether neutralizing antibodies or small-molecule inhibitors of IL-31RA can alter muscarinic signaling in ASMC. If so, therapeutic efficacy might be improved with these antibodies and new inhibitors, which may reduce the expression of IL-31RA and/or muscarinic signaling in ASMC. However, even highly effective anti-AHR therapies sufficient to inhibit IL-31RA-driven contraction of ASMC may need to be combined with therapeutics that mitigate inflammation and goblet cell hyperplasia.
In summary, we have described the role of IL-31RA in the induction of AHR, with limited effects on airway inflammation and goblet cell hyperplasia, and this role is independent of its ligand IL-31. Importantly, we identified a novel mechanism underlying ASMC contractility that involves IL-31RA dependent increases in CHRM3-driven calcium signaling and phosphorylation of MLC. Together, these results suggest an important role for IL-31RA in the regulation of AHR and identifying the molecular events underlying IL-31RA-driven AHR may lead to the development of novel therapeutic approaches against AHR in allergic asthma. Lungs were sliced into 300 µm section in 4 °C cold HBSS without calcium using a VF-310Z vibratome (Pecisionary Instruments, Winchester, MA, USA). PCLS were collected in DMEM media without serum, and the media were changed at least four times with intermittent shaking to remove residual agarose and incubated in a humidified incubator with 5% CO2 at 37 °C. On the following day, the media was replaced with fresh media without serum for the same-day experiment. For cytokine treatments, PCLS were treated with IL-13 (50 ng/mL, R&D Systems) or IL-31 (500 ng/mL, R&D Systems) for 24 h in a low-serum media (1% FBS) prior to the contraction assay using increasing doses of MCh. Lung slices were allowed to rest and relax before the next dose of MCh was administered to the samples. Changes in the airway lumen area were recorded in a time-lapse setting with images captured every 5 s for 5 min following each dose of MCh using a 10x objective on a temperature-controlled Nikon inverted microscope at 37 °C with 5% CO2 (NIKON Ti2 widefield, Japan). Airway areas were analyzed using NIKON NIS-Elements analysis software, and changes were expressed as the percentage area of contraction compared to the initial baseline area.

RNA isolation and real-time PCR.
Lung tissues were dissociated using TRIzol (Life Technologies) using beads and a high-speed homogenizer (Thermo Fisher). RNA was isolated from lung tissues, primary mouse ASMs, or primary asthmatic human bronchial ASMC (Lonza, Walkerville,MD, USA) using an RNAeasy mini kit (QIAGEN) following the manufacturer's instructions and as previously described in our previous studies 28,72 . cDNA was synthesized using Superscript III (ThermoFisher), and real-time PCR was performed using  (Table S1).
Collagen gel contraction assay. Human bronchial smooth muscle cells were obtained from Lonza (Wakersville, MD, USA), and murine ASMC were prepared via enzymatic digestion of the mouse trachea at 37 °C for 60 min, as previously described 73 . A single-cell suspension of digested trachea was seeded on a 100 mm petri dish (n = 4/dish) using Hams/F12 culture media supplemented with 10% FBS and antibiotics. Spindle-shaped cells were allowed to grow to 80% confluence, and cells from passages 1-2 were used for the collagen gel contraction assay.
ASMCs were seeded into rat tail collagen gel matrices as described previously 74,75 . To determine the effect of cytokines on ASMC contraction, cells were treated with cytokines (rIL-31 500 ng/mL or rIL-13, 50 ng/mL) or media for 48 h prior to embedding them in collagen gel.
Collagen gels were prepared using cells treated with cytokines and media and grown under similar conditions in Ham's/F12 complete media. The collagen gels were detached from the walls, and images were captured at 0 ,12 or 16, 24, and 48 h using a stereo microscope. The area of contraction was measured using ImageJ software and described as the percentage of the contracted area compared with the baseline area of the gel.        Data are shown as the mean ± SEM (n = 3/group). One-way ANOVA was used; *P < 0.05.